In the United States urothelial carcinoma (UC) of the bladder is the 4th most frequent malignancy in men, with around 80,500 new cases and 17,500 deaths in 2019. Despite being the most costly cancer to treat over a patient's lifetime, bladder cancer remains underfunded. High-grade (HG), muscle invasive bladder tumors account for the majority of these deaths as patients with metastatic disease have a 5-year survival rate of only 15%. The current standard first line therapy for metastatic disease is cisplatin-based combination chemotherapy or immune checkpoint inhibition in patients who are platinum ineligible. Novel immune checkpoint (IC) inhibitors, including anti-PD1 and anti-PD-L1, represent a paradigm shift in cancer therapy in patients with advanced UC however, only 15-25% of patients treated in published trials had objective responses to single agent IC inhibition. In patients with clinically localized, muscle-invasive bladder cancer (MIBC), there is a high level of evidence to support the use of cisplatin-based neoadjuvant chemotherapy. Two widely accepted regimens with therapeutic equipoise are MVAC (methotrexate, vincristine, adriamycin, and cisplatin) and GC (gemcitabine/cisplatin). While trials in advanced bladder cancer therapy are evaluating the combination of chemo and immunotherapy, to rationally combine these two therapeutic modalities it is imperative to precisely understand how MVAC and GC impact the immune microenvironment and how to best sequence chemo and immunotherapy. Preliminary studies from the Kim and Vincent laboratories show that MVAC and GC have differing effects on the immune microenvironment in bladder cancers of the luminal molecular subtype. Tumors of the luminal subtype, which have low baseline immune infiltration have a significant increase in immune gene signature expression and restriction of tumor-specific T cell receptor (TCR) clonotypes in the peripheral blood after MVAC but not GC treatment. In contrast, GC significantly increases gene signatures known to promote resistance to IC therapy in bladder cancer (Fibroblast TGFB Response Signature [FTBRS] and EMT_Stroma). These results in aggregate suggest that in luminal bladder cancers, MVAC promotes an inflamed tumor immune microenvironment permissive to IC inhibition, while GC increases stromal expansion, known to correlate with immunotherapy resistance. In aggregate our preliminary data suggest that MVAC should combine better with IC therapy than GC. Successful completion of this proposal will validate these findings in a prospective cohort of paired pre and post MVAC or GC samples collected in the context of a randomized clinical trial (SWOG1314), determine which specific anti-neoplastic agents alter the immune microenvironment and promote sensitivity to immunotherapy, as well as outline how to best sequence anti-neoplastic agents and immune checkpoint blockade in urothelial bladder cancer.